Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Tips and tricks for successfully culturing and adapting human induced pluripotent stem cells

doi: 10.1016/j.omtm.2021.10.013

Figure Lengend Snippet: Generation of feeder cells (A) Scheme representing the followed workflow to generate the stock of feeder cells. Scheme prepared with BioRender. (B) Histogram representing the results of the cell-cycle analysis by flow cytometry. Irradiation of the foreskin fibroblast cell line HFF-1 at 75 Gy (green line) caused cell-cycle arrest (arrow), in comparison with unirradiated HFF-1 cells (purple area). (C) Homogeneous monolayer of feeder cells at >80% confluency. Scale bar, 200 μm.

Article Snippet: • Basic fibroblast growth factor (bFGF) (13256029, Gibco Thermo Fisher Scientific) • Bovine serum albumin (BSA) 9048-46-8, Sigma) • Dimethyl sulfoxide (DMSO) (67-68-5, Sigma-Aldrich Química SA) • Dulbecco's modified Eagle's medium (DMEM) (12-604F, Lonza) • DMEM knockout without L-glutamine (KO-DMEM); (10829018, Gibco Thermo Fisher Scientific) • DPBS with calcium and magnesium (14040133, Gibco Thermo Fisher Scientific) • Fluorescence-activated cell sorting buffer (Becton Dickinson) • Fetal bovine serum (FBS) (10082147, Gibco Thermo Fisher Scientific) • Gentle cell dissociation reagent (07174, STEMCELL Technologies) • Geltrex hESC-qualified, ready-to-use, reduced growth factor basement membrane matrix (A1569601, Gibco) • GlutaMAX 100X (35050061, Gibco Thermo Fisher Scientific) • Human foreskin fibroblasts, HFF-1 cell line (CRL2429, ATCC) • Iscove’s modified Dulbecco medium (IMDM) (12440053; Gibco Thermo Fisher Scientific) • Knockout serum replacement (10828028, Gibco Thermo Fisher Scientific) • 2-Mercaptoethanol (50 mM; 31350010, Gibco Thermo Fisher Scientific) • Matrigel basement membrane matrix (354234, Corning) • Non-essential amino acids 100X (11140050, Gibco Thermo Fisher Scientific) • Phosphate-buffered saline tablets (BR0014G, Oxoid) • Phosphate-buffered saline with Ca 2+ and Mg (14287, Thermo Fisher Scientific) • Penicillin/streptomycin (P/S) (15140122, Gibco Thermo Fisher Scientific) • Propidium iodide (PI) (P4170, Sigma-Aldrich) • rh-Laminin-521 521 (A29249; Thermo Fisher Scientific) • RNAse (500 μg/mL; Roche) • RevitaCell 100X (A2644501; Gibco) • ROCK inhibitor Y-27632 (04-0012, Stemgent) • Saline serum (154 mmol/L Na and 154 mmol/L Cl – , Fresenius Kabi) • StemFlex basal medium (A3349301, Gibco Thermo Fisher Scientific) • StemFlex Supplement 10X (A3349201, Gibco Thermo Fisher Scientific) • Trypan blue staining (T8154-20 ML, Sigma) • TrypLE Select Enzyme (1X), no phenol red (12563011, Gibco Thermo Fisher Scientific) • Versene solution (15040066, Gibco) • 0.05% trypsin-EDTA (25300054, Gibco) • 0.25% trypsin-EDTA (25200056, Gibco)

Techniques: Cell Cycle Assay, Flow Cytometry, Irradiation